From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Regular Article
CLINICAL TRIALS AND OBSERVATIONS
Biomarkers of terminal complement activation confirm the diagnosis of
aHUS and differentiate aHUS from TTP
Spero R. Cataland,1 V. Michael Holers,2 Susan Geyer,1 Shangbin Yang,3 and Haifeng M. Wu3
1
Internal Medicine, Ohio State University, Columbus, OH; 2Medicine, University of Colorado, Aurora, CO; and 3Department of Pathology, Ohio State
University, Columbus, OH
Atypical hemolytic uremic syndrome (aHUS) is characterized by dysregulated complement activity, the development of a thrombotic microangiopathy (TMA), and widespread
end organ injury. aHUS remains a clinical diagnosis without an objective laboratory test
• Biomarkers of complement
to confirm the diagnosis. We performed a retrospective analysis of 103 patients enrolled
activation can confirm the
in the Ohio State University TTP/aHUS Registry presenting with an acute TMA. Nineteen
diagnosis of aHUS.
• Complement biomarkers may patients were clinically9 categorized as aHUS based on the following criteria: (1)
platelet count <100 3 10 /L, (2) serum creatinine >2.25 mg/dL, and (3) a disintegrin and
be useful to differentiate
metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) activity >10%.
aHUS from TTP.
Sixteen of 19 patients were treated with plasma exchange (PEX) therapy, with 6/16 (38%)
responding to PEX. Nine patients were treated with eculizumab with 7/9 (78%) responding to therapy. In contrast to thrombotic
thrombocytopenic purpura (TTP) patients, no aHUS patients demonstrated ultralarge von Willebrand factor multimers at
presentation. Median markers of generalized complement activation (C3a), alternative pathway (Bb), classical/lectin pathway (C4d),
and terminal complement activation (C5a and C5b-9) were increased in the plasma of these 19 patients. Compared with a cohort of
ADAMTS13-deficient TTP patients (n 5 38), C5a and C5-9 were significantly higher in the 19 patients clinically characterized as aHUS,
suggesting that pretreatment measurements of complement biomarkers C5a and C5b-9 may confirm the diagnosis of aHUS and
differentiate it from TTP. (Blood. 2014;123(24):3733-3738)
Key Points
Introduction
Patients and methods
Significant advances in the treatment of atypical hemolytic uremic
syndrome (aHUS) have placed an increased emphasis on the rapid and
accurate differentiation of aHUS from acquired thrombotic thrombocytopenic purpura (TTP).1 Although both disorders share the common end
point of widespread microvascular disease and end organ injury, their
distinct underlying mechanisms of microvascular injury likely explain
their differing responses to plasma exchange (PEX) and the response of
patients with aHUS to complement inhibition therapy. Despite
advances in our understanding of both aHUS and acquired TTP
in recent years, both diseases remain clinical diagnoses.
Although severely deficient a disintegrin and metalloprotease
with thrombospondin type 1 motif, 13 (ADAMTS13) activity can
confirm the clinical diagnosis of acquired TTP, no such objective
single test is available to confirm the clinical diagnosis of aHUS. This
is especially problematic because aHUS is a condition that often
requires prolonged complement inhibition therapy.1,2 The availability
of an objective biomarker to confirm the clinical diagnosis of aHUS
would be invaluable to clinicians who must diagnose and treat these
challenging patients. In addition, a surrogate biomarker that could
predict a response to complement inhibition early in the course of
therapy would be a significant advance over our present method of
using biomarkers of hemolysis and recovery of end organ function, a
process that may take several weeks before any objective response
to therapy is appreciated clinically.1,3
We performed a retrospective study of all patients enrolled into the Ohio
State University TTP/aHUS Registry since its inception in 2003. This
registry is composed of patients that present or are referred to our institution
with an acute thrombotic microangiopathy (TMA). All patient data reported
in this manuscript are from patients that have been enrolled in this
Institutional Review Board-approved registry. This study was conducted
in accordance with the Declaration of Helsinki. Beginning with a total of
103 enrolled patients, 76 patients were excluded from this analysis because of
ADAMTS13 activity ,10%, 3 patients were excluded because of their
subacute presentation (platelet count .100 3 109 /L), with an additional
5 patients excluded because a pretreatment plasma sample was not
available (Figure 1). From this cohort of acute TMA patients, 19 patients
with a platelet count ,100 3 109/L, serum creatinine .2.25 mg/dL, and
measurable ADAMTS13 activity (.10%) were selected for study. Although
there is no objective diagnostic test that defines the diagnosis of aHUS, it has
been hypothesized that these criteria identify a cohort of patients that is
likely to be enriched for the diagnosis of aHUS.4,5 The clinical and
demographic data for all 19 patients are shown in Table 1.6 This was the
initial presentation for 17/19 (89%) patients, with 16/19 patients undergoing PEX therapy at the time of presentation.
The criteria to define a response to therapy in this study included
a normal platelet count and a stable or improved serum creatinine (at 6
months of follow-up) independent of PEX therapy or with continued
therapy with eculizumab (Soliris). In patients presenting after September
Submitted December 31, 2013; accepted March 25, 2014. Prepublished online
as Blood First Edition paper, April 2, 2014; DOI 10.1182/blood-2013-12547067.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2014 by The American Society of Hematology
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
3733
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3734
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
CATALAND et al
Figure 1. Criteria used to select patients with aHUS
for analysis and their response to PEX therapy and
eculizumab.
2011 when eculizumab received regulatory approval from the US Food
and Drug Administration, eculizumab was available for patients with a
clinical diagnosis of aHUS. The criteria used by our group7 and others2 to
define the failure of PEX therapy and to initiate therapy with eculizumab
for a presumed diagnosis of aHUS include the following: (1) the failure
to achieve a hematologic response (improvement in platelet count and
decrease in the LDH) over the first 4-5 days; (2) progressive end organ
injury (renal and/or neurologic) over the first 4-5 days of PEX therapy;
and (3) nondeficient (.10%) ADAMTS13 activity. These criteria were
used as evidence for the failure of PEX and the presumptive diagnosis of
aHUS; in these cases, PEX was discontinued and therapy with eculizumab
begun where it was available to patients. Follow-up for all TMA patients
is standardized at our institution, with all patients being seen weekly for
the first 4 weeks after discharge from the hospital, then monthly for the
next 5 months, and then every 3 months longitudinally.
Complement biomarker assay methodology
Complement biomarker studies were performed on banked plasma samples
at presentation, prior to the initiation of either PEX or eculizumab. Three
patients underwent dialysis prior to samples being obtained, but there was
no significant difference between these samples obtained after dialysis and
those obtained from the remaining patients with samples obtained before
undergoing hemodialysis (supplemental Table 4, available on the Blood
Web site). In some patients, additional samples both during PEX and during
long-term follow-up were available for study, but not at regular, predefined
Table 1. Demographic and clinical features of the 19 subjects clinically characterized as aHUS compared with a cohort of 38 patients with
TTP with severely deficient ADAMTS13 activity
Median demographic and clinical data at presentation
Age
(range)
aHUS patients (n 5 19)
TTP patients (n 5 38)
Sex
(M/F)
Race
(AA/W)
Dialysis
47 (20-69)
3/16
3/16
14/19 (74%)
42.5 (18-76)
12/26
8/30
0/38
AA, African American; F, female; LDH, lactate dehydrogenase M, male; W, white.
Platelets
(150-400 3 109)
Creatinine
(<1.1 mg/dL)
LDH (<190 U/L)
C3
(970-1576 mg/L)
46 (8-93)
4.39 (2.38-12.61)
1094 (140-3548)
915 (561-1550)
12 (2-45)
1.15 (0.65-4.11)
966 (243-2449)
―
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
COMPLEMENT BIOMARKERS AND DIAGNOSIS OF aHUS
3735
time points. Uniform banking and processing procedures for all banked
samples have been in place since the initiation of our tissue bank in 2003,
minimizing the potential impact of handling and storage on the results
obtained. Complement proteins were chosen to study the pathway of
complement activation and included the following: C4d (classical and lectin),
factor Bb (alternative), generalized complement activation (C3a), and
terminal complement activation (C5a and C5b-9).
Complement studies were performed using commercial enzyme-linked
immunosorbent assay kits as reported previously.6 The assay kits for C4d,
factor Bb, C3a, and C5b-9 were from Quidel (San Diego, CA). The assay kit
for C5a was from BD (Franklin Lakes, NJ). The reference range for each of
the complement assays was established using 40 local healthy donors.
Assay validation studies indicated that complement factor Bb, C5a,
and C5b-9 are very stable analytes. The assay results were not significantly
affected by sample storage at room temperature for 2 hours or overnight in
a refrigerator prior to processing, nor were they affected by 1 cycle of sample
freeze/thaw. C4d was also a relatively stable analyte, but the results were 30%
higher when the blood was stored overnight in a 4°C refrigerator before
processing. C3a was the least stable analyte with extended storage and freeze/
thaw cycles giving rise to a result that was .50% higher than the result from
a freshly banked sample without experiencing a freeze/thaw cycle. Given that all
samples in this study have been banked and processed in a uniform manner,
these potential sources of error/variability are less relevant to these data.
Nonetheless, these issues must be considered when comparing the results of
C4d and C3a studies from different laboratories, and our data support the use of
Bb, C5a, and C5b-9 as more reliable clinical assays of complement activation.
ADAMTS13 assay methodology
ADAMTS13 activity was measured using a surface-enhanced laser
desorption/ionization time-of-flight mass spectrometer as described
previously.8 ADAMST13 inhibitor titer was determined using the same
technology and calculated as a dilution of patient plasma that neutralizes 50%
ADAMTS13 activity in pooled normal plasma. ADAMTS13 immunoglobulin G
levels were determined using a commercial kit from American Diagnostica
as reported previously.8
von Willebrand factor (VWF) multimer analysis
Citrated plasma from each patient was diluted 1:40 in 70 mM tris
(hydroxymethyl)aminomethane-HCl (pH: 8.8) containing 4 mM EDTA,
2.4% sodium dodecyl sulfate, 0.67 M urea, 0.1% bromophenol blue, and 5%
glycerol and incubated in a 60°C water bath for 20 minutes. A 5-mL sample
was then electrophoresed on a 1.6%, vertical, discontinuous agarose gel
(11 3 15 cm) at a constant current of 4 mA for 18 hours. After the run,
proteins were transferred to a polyvinylidene fluoride membrane at
a constant current of 500 mA for 4 hours. After blocking the membrane
with 5% dry milk solution, immobilized proteins were reacted with rabbit
anti-human VWF polyclonal antibody (1:2500 dilution; Dako) for 2 hours,
followed by application of the HyGLO Chemiluminescent HRP Antibody
Detection Kit (Denville Scientific). The immunoreactive bands of VWF
multimers on the film were scanned by densitometry to get images for
analysis. In most of the experiments, normal control plasma showed 15-17
distinct immunoreactive bands. Similar numbers of immunoreactive bands
were seen when 40 normal, healthy donors were evaluated. In order to more
objectively define the fraction best representing ultralarge VWF (ULVWF),
we grouped the large VWF multimers together, with the area higher than band
#15 indicated by an arrow, and collectively referred to as ULVWF (Figure 2).
Complement protein mutation analysis
The reported complement protein mutation studies were performed by
a reference laboratory at the University of Iowa.
Statistical methods
A general linear model was used for comparison of the complement biomarkers in terms of the presence of mutations, response to PEX, and the
comparison of the aHUS cohort with the cohort of acquired TTP. Race,
Figure 2. VWF multimeric analysis of pretreatment samples for TTP and aHUS
patients. In each run shown, a normal control plasma (NC) and an abnormal control
plasma (AC, obtained from a patient with type 2 von Willebrand disease) were
included to verify the performance and resolution of the analysis. The area above the
solid line in the figure represents the larger multimers with molecular weight higher
than band #15 (indicated by an arrow) counted from the bottom of the gel. This area,
indicated by an vertical line in the figure, is referred to as ULVWF. (A) Multimeric
analysis from 19 acquired TTP patients. (B) Multimeric analysis from 19 aHUS
patients. A sample from a TTP patient containing ULVWF is included in panel B for
comparison. In a few aHUS patients, faint bands above the dotted line can be seen
that represent normal variation rather than pathologically increased ULVWF multimers.
gender, and age were used as covariates. Where necessary, data sets were log
transformed to fit a normal distribution. If after log transformation the data set
still did not fit a normal distribution, an independent Mann-Whitney U test
was used for comparison using age, sex, and race as covariables. The SPSS
program was used for all analyses. All calculated P values were 2-sided with
the level of significance set at P , .05.
Results
A total of 19 patients were included in this analysis. The demographic features and the clinical laboratory data at presentation are
shown in Table 1. Three of the 19 (16%) patients presented immediately postpartum, and 4/19 (21%) had previously undergone
kidney transplantation. The finding that 7/19 aHUS patients presented postpartum and post–kidney transplantation is not surprising
given that these 2 scenarios are common clinical presentations of
aHUS. PEX was started in 16/19 (84%) of patients at the time of their
initial presentation. Three patients were not treated with PEX therapy
based on their subacute presentations, which were judged to be atypical for an acute TTP episode by the treating physician, and were
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3736
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
CATALAND et al
Table 2. Complement biomarker studies at presentation for all subjects and in the context of mutation status and response to PEX
Median complement biomarkers (ng/mL) (normal range)
All patients (n 5 19)
Factor Bb (244.3-960.8)
C4d (278.5-1845.9)
C5b-9 (33.9-238.2)
C5a (18.6-47.9)
C3a (6.9-242.3)
7386 (603-30 614)
2914 (1394-15 210)
1098 (422-4840)
115 (55-280)
1237 (79-13 726)
1353 (79-3490)
Mutation present* (n 5 6)
9299 (1050-30 614)
2571 (1640-3831)
1480 (422-4840)
125 (56-269)
No mutation (n 5 6)
1756 (910-15 096)
3338 (1394-4981)
954 (621-1317)
97 (55-280)
.206
.795
.189
P value
PEX response (n 5 6)
PEX nonresponder (n 5 10)
P value
.322
.401
759 (566-1391)
14 945 (7386-20 443)
3495 (1640-4680)
1394 (1024-1920)
141 (105-214)
1266 (590-13 726)
3103 (603-30 614)
3066 (1394-4981)
1063 (422-4840)
108 (55-269)
1158 (79-3834)
.593
.675
.185
.818
.389
Twelve of the 19 subjects had mutation studies performed.
*Mutations studied included the following: factor H, factor I, factor B, C3, membrane cofactor protein (MCP), thrombomodulin, and CFHR3-CFHR1.
treated initially with eculizumab. Renal failure requiring dialysis was
present in 13/16 (81%) of patients treated with PEX. In the 3 patients
that were not treated with PEX, acute renal injury was present in all
3 patients (serum creatinine .3 mg/dL), but only one-third of these
patients required dialysis at presentation.
Response to PEX
Six of 16 patients (38%) met the criteria for a response to PEX
therapy, including independence from dialysis in the 4 patients that
required dialysis at presentation. The median number of PEX procedures to achieve a normal platelet count in these 6 patients was 11.5
(range, 5-32). Ten patients did not respond to PEX after a median of 6
PEX procedures (range, 4-13). Two nonresponding patients died 3
weeks and 3 months after their initial presentations secondary to acute
respiratory failure and a massive hemispheric infarct, respectively.
In the 8 remaining patients that failed PEX, 6 discontinued PEX
therapy and immediately started therapy with eculizumab. The 2
remaining patients had PEX discontinued and were discharged with
a persistent thrombocytopenia and end-stage renal disease requiring
hemodialysis. Eculizumab was not available for these 2 patients
because their presentation occurred prior to the regulatory approval
of eculizumab for the treatment of aHUS.
Response to eculizumab
A total of 9 patients were treated with eculizumab, including the 3
patients who received eculizumab without previously being treated
with PEX. Hematologic responses (normalization of the platelet
count) occurred in 7/9 (78%); 8/9 (89%) patients had improvements in renal function after therapy with eculizumab. Six of the 9
eculizumab-treated patients were dialysis dependent at the start of
therapy, with all 6 becoming independent of the need for dialysis.
Two patients did not normalize their platelet count after 8 and
10 months of therapy, respectively. The first patient did, however,
recover renal function and became independent of dialysis despite
the lack of a hematologic response. The second patient never
showed any significant improvement in renal function on therapy
with eculizumab and started hemodialysis 10 months after starting
therapy with eculizumab. This patient was found to have heterozygous mutations of both complement factor (CF)I and CF
H-related genes 3 and 1 (CFHR3-CFHR1), but testing for CFH
autoantibodies was not performed. Significant renal injury was
present in this patient at the time of presentation (serum creatinine
.3 mg/dL, estimated glomerular filtration rate ,15 mL/minutes
per m2) that had been present for at least 3 months prior to her
presentation at our institution. No other extrarenal manifestations
attributable to aHUS were found in this patient.
Complement biomarker studies
The median complement biomarker data for all 19 subjects prior to
the initiation of therapy are shown for all 19 patients in Table 2.
Activation of complement as defined by an increase in both C3a and
C5a was seen in 18/19 (95%) of patients. More pronounced activation of the alternative pathway (factor Bb) and the terminal complement pathway (C5b-9) was seen in this cohort of 19 patients.
Sixteen of 19 (84%) patients had increased levels of factor Bb at
presentation, and increased levels of C5b-9 were seen in all 19
patients. The complement biomarker measuring activation of the
classic complement pathway (C4d) was increased (15/19 [79%]), but
to a lesser degree than to what was seen in the alternative and terminal
pathways. All patients that were treated with either PEX or eculizumab
therapy had increased pretreatment biomarkers of terminal complement activation (C5a and C5b-9).
We compared the pretreatment complement biomarkers from this
cohort of 19 patients clinical diagnosed with aHUS with a previously
published cohort of 38 TTP patients enrolled in the Ohio State TTP/
aHUS Registry but characterized by severely deficient ADAMTS13
activity.6 In comparing these 38 acquired TTP patients to this cohort
of 19 patients, pretreatment levels of C5a, C3a, and C5b-9 were
significantly higher in the cohort of 19 patients clinically diagnosed
with aHUS compared with published data from the 38 patients
with TTP characterized by severely deficient ADAMTS13 activity
(Table 3). Although the median and mean complement biomarkers
were significantly different between these groups, there was still
overlap in these markers between the diagnosis groups. A clear
cutoff to differentiate one group vs the other based on these markers
was not readily apparent in these data, even when evaluating the logtransformed levels of the markers (supplemental Figure 3A-E).
Complement mutation studies
Twelve patients were studied for the presence of mutations (factor H,
factor I, factor B, C3, MCP, thrombomodulin, and CFHR3-CFHR1)
of complement proteins, with 6/12 having documented heterozygous
mutations (CFH, 2; complement factor I, 2; C3, 1; MCP, 1; and CFHR3CFHR1, 1) and 1 patient found to have 2 mutations. There was no
significant difference in any of the complement biomarkers studied
between those with and without documented mutations (Table 2).
Similarly, there was no significant difference in these same pretreatment
biomarkers in the 6 patients that responded to PEX compared with
the 10 PEX nonresponders. Eight of the 9 eculizumab-treated
patients had mutation studies performed, with 5/8 (63%) found to
have one of the above-mentioned mutations. Six of the 7 eculizumabresponding patients had mutation studies performed, with 4/6
responders having a documented complement protein mutation.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
COMPLEMENT BIOMARKERS AND DIAGNOSIS OF aHUS
3737
Table 3. Comparison of pretreatment complement biomarkers obtained from 19 patients with clinically diagnosed aHUS vs a cohort of
38 patients with ADAMTS13-deficient TTP
Complement biomarkers (ng/mL)
Clinical diagnosis
Factor Bb (244.3-960.8)
C4d (278.5-1845.9)
C5b-9 (33.9-238.2)
C5a (18.6-47.9)
585 (210-1924)
75 (29-210)
777 (128-4782)
2914 (1394-15 210)
1098 (422-4840)
115 (55-280)
1237 (79-13 730)
.706
< .0001
.004
.031
Acquired TTP*6 (n 5 38)
2153 (343-5448)
3534 (458-7450)
aHUS* (n 5 19)
7386 (603-30 610)
.063
P value†
C3a (6.9-242.3)
Compared with the acquired TTP patients, patients clinically diagnosed aHUS tended to have significantly higher C5b-9 and C5a levels. P values reflect comparisons of
complement biomarkers between diagnosis groups using Wilcoxon rank sum tests. Bolded text signifies the values that were statistically significant at the P , .05 level.
*For the acquired TTP patients, the mean data for each complement biomarker are shown; whereas for the smaller aHUS cohort, the median biomarker data are
presented.
†Compared with the 19 clinically diagnosed aHUS patients. The Mann-Whitney independent U test was used for the Bb and C4d comparisons, whereas a generalized
linear model was used to compare C5b-9, C5a, and C3a, using age, sex, and race as covariables.
VWF multimer analysis
In this study, the VWF multimers present at the time of acute presentation were compared between patients clinically characterized as
aHUS and a cohort of ADAMTS13-deficient TTP patients. Samples
studied included the pretreatment samples from the 19 patients
clinically diagnosed with aHUS in this report and pretreatment
samples from 19 TTP patients randomly selected from patients
enrolled in our registry. As shown in Figure 2A, at least 10 of the 19
TTP patients demonstrated a significant accumulation of ULVWF
at the time of acute clinical presentation. In contrast, as shown in
Figure 2B, all 19 patients with a diagnosis of aHUS did not show
evidence for any significant accumulation of ULVWF multimers.
Discussion
The reported efficacy and regulatory approval of eculizumab for the
treatment of aHUS have emphasized the need for more objective
diagnostic testing to confirm the diagnosis of aHUS and differentiate
it from acquired TTP.1 Although the presence of severely deficient
ADAMTS13 activity confirms the diagnosis of acquired TTP, no
single such biomarker exists to confirm the diagnosis in patients with
a suspected diagnosis of aHUS. Such a biomarker would be an
important confirmatory test for the clinical diagnosis of aHUS given
both the expense of therapy with eculizumab as well as the presumed
need for long-term therapy.2 Complement biomarkers documenting
activation of the alternative pathway and terminal pathway activation
at the time of presentation would also eliminate the complete reliance
on the response to complement inhibition therapy to confirm the
clinical diagnosis of aHUS, which can take several weeks in the case
of renal injury. In addition, in a patient clinically suspected to have
aHUS, the finding of normal biomarkers of terminal complement
activation (C5a and C5b-9) would argue against the role of dysregulated
complement activity as the etiology of a patient’s TMA findings.
One limitation in the development of objective diagnostic testing
for the diagnosis of aHUS is the dependency on clinical and laboratory criteria to define the diagnosis for study. Although aHUS
remains a clinical diagnosis, there is mounting data that the
ADAMTS13 activity, the extent of renal injury at presentation, and
the response to PEX can be used collectively to define a cohort
of patients consistent with the diagnosis of aHUS.2,5,7 These criteria
are presently used by our group to prospectively identify patients
consistent with the diagnosis of aHUS and were used to identify
patients for this study.7 The accuracy of our classification cannot be
absolutely confirmed, but the finding of complement mutations in
half of the studied patients consistent with previously published
studies2,9,10 and the high rates of response to therapy in the eculizumabtreated patients support the hypothesis that this cohort of patients
is accurately classified as aHUS. In addition, postpartum and
post–kidney transplantation are common clinical scenarios associated
with the diagnosis of aHUS. The finding of 7 of 19 patients presenting
in the immediate postpartum state and postkidney transplantation
(given the high rates of recurrence after kidney transplantation) would
further support the clinical diagnosis of aHUS.10
Given the recent clinical availability of eculizumab, patients were
more likely to be categorized as nonresponders to PEX after a fewer
number of PEX procedures, resulting in more recently presenting
patients being classified as nonresponders after a fewer number of
PEX procedures compared with those presenting prior to the availability
of eculizumab. Seven of 10 patients in the PEX nonresponding cohort
had the option of going on to therapy with eculizumab after failing PEX,
explaining in part the earlier clinical decision to discontinue PEX and
initiate therapy with eculizumab. It is plausible that the PEX
nonresponding patients could have recovered after a more prolonged
course of PEX similar to the longer PEX treatments seen in the PEXresponding patients. The question could also be raised as to whether
these 6 patients that responded to PEX therapy might also have
responded to supportive care equally. One of the 6 PEX-responding
patients had complement mutation studies performed that demonstrated a mutation of MCP, a mutation that predicts a more benign
course and identifies patients that may also recover with supportive
care alone. These issues limit the application of these data to accurately define the response rates to PEX in patients with clinically
diagnosed aHUS in this study.
Although pathological activation of the alternative pathway and
dysregulated complement activity are central to the pathophysiology
of aHUS, complement activation has also been reported in patients
with ADAMTS13-deficient TTP.6,11 Réti et al11 reported the finding
of increased levels of C3a and C5b-9 in 23 patients with ADAMTS13deficient TTP, indicative of generalized and terminal complement
(membrane attack complex formation), respectively. Similar data have
also been published by our group showing complement activation at
presentation in 38 patients with ADAMTS13-deficient TTP.6 In this
report, 33/38 (87%) patients had evidence for complement activation
as defined by increases in both C3a and C5a. Although activation of
complement is seen in both disorders, we hypothesize that terminal
complement activation as measured by C5a and C5b-9 may be useful
to objectively confirm the clinical diagnosis of aHUS and differentiate
it from acquired TTP. At presentation, more significant elevations of
C5a and C5b-9 and nonseverely deficient ADAMTS13 activity in the
context of a patient responding poorly to PEX may collectively be
useful to differentiate aHUS from acquired TTP. Additionally, measurement of terminal complement activation in TTP patients as measured by C5a and C5b-9 might also be useful to identify patients
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3738
BLOOD, 12 JUNE 2014 x VOLUME 123, NUMBER 24
CATALAND et al
at greater risk of death during that acute TMA episode as reported
previously.6
It could also be hypothesized that these same complement
biomarkers might find utility as a biomarker to predict the response
therapy with eculizumab. Support for the potential utility of these
complement biomarkers as markers of response to therapy can also
be found in the additional plasma samples that were available from
5 of 9 eculizumab-treated patients during follow-up at varied time points
(1.5 weeks to 16 months) during continuous eculizumab therapy. In
these 5 patients that were recovering clinically on eculizumab therapy,
there was a trend showing decreased levels of C5b-9 in patients
clinically responding to therapy with eculizumab with variability in
C5a levels during ongoing therapy with eculizumab (data not shown).
Although it would be predicted that both C5a and C5b-9 should both
decline after therapy with eculizumab, the follow-up samples were
obtained on the day that they were due for their next dose of
eculizumab, at the nadir of the anti-C5 antibody levels, providing
one possible explanation for lack of a clear decline in posttreatment C5a levels. Although the limited number of samples obtained
at varying time points during their course of therapy precludes any
definite conclusions from being drawn, these data should serve as
hypothesis-generating data for future prospective studies.
It is important to mention the potential confounding effects that
can be seen with differing methods of handling and processing of
clinical samples for complement biomarker studies.12 As described
previously, we found C3a to be the least stable analyte of the complement biomarkers studied, with differing storage times and freezethaw cycles markedly affecting the results obtained. In contrast, C5a
and C5b-9 assays performed well across many conditions, providing
reliable and reproducible results that make them the most attractive
complement biomarkers for routine clinical study.
The data from VWF multimer analysis confirm the work from
previous publications that the formation of ULVWF is associated
with clinical development of acute TTP.13,14 Additionally, our data
confirm previous work that ULVWF multimers are not abnormally
accumulated in all cases of acquired TTP at the time of acute
presentation.13 Conceivably, an early presentation of disease may
exhibit the accumulation of ULVWF in the blood, whereas a fulminant
TTP case may not demonstrate the presence of ULVWF multimers
because of consumption related to the development of extensive
platelet thromboses. Future studies using a larger TTP cohort with
longitudinal sampling may allow for a more detailed study of the
relationship between severity of ULVWF accumulation, laboratory data, and clinical outcome, in the context of the measured
ADAMTS13 activity. However, importantly for this study, we
demonstrate for the first time that the abnormal accumulation of
ULVWF is not present in a cohort of clinically diagnosed aHUS
patients at the time of acute presentation. This novel observation
strongly suggests that in contrast to TTP, VWF-mediated platelet
thrombosis is not a primary mechanism for development of TMA
in patients with aHUS.
In summary, these data suggest that the use of these biomarkers of
complement activation, specifically C5a and C5b-9, may be useful
clinically to confirm the clinical diagnosis of aHUS and differentiate
it from acquired TTP. Although limited by the retrospective nature of
the data, they should form the basis for future studies to evaluate
their role in the confirmation of the diagnosis as well as a tool to
objectively monitor the response to complement inhibition therapy.
Acknowledgment
This work was supported in part by a US Food and Drug Administration government grant (R01 FD003932).
Authorship
Contribution: S.R.C. designed the research, analyzed the data,
and wrote the manuscript; V.M.H. analyzed the data and edited
the manuscript; S.G. performed the statistical analysis of the data;
S.Y. performed the experiments, analyzed the data, and edited
the manuscript; and H.M.W. designed the research, analyzed the
data, and wrote/edited the manuscript.
Conflict-of-interest disclosure: S.R.C. and H.M.W. are consultants to and received research funding from Alexion. V.M.H is
a consultant to Alexion. The remaining authors declare no competing financial interests.
Correspondence: Spero R. Cataland, Department of Internal
Medicine, Ohio State University, A361 Starling Loving Hall,
320 W 10th Ave Columbus, OH 43210; e-mail: spero.cataland@
osumc.edu.
References
1. Legendre CM, Licht C, Muus P, et al. Terminal
complement inhibitor eculizumab in atypical
hemolytic-uremic syndrome. N Engl J Med. 2013;
368(23):2169-2181.
2. Zuber J, Fakhouri F, Roumenina LT, Loirat C,
Frémeaux-Bacchi V; French Study Group for
aHUS/C3G. Use of eculizumab for atypical
haemolytic uraemic syndrome and C3
glomerulopathies. Nat Rev Nephrol. 2012;8(11):
643-657.
3. Salem G, Flynn JM, Cataland SR. Profound
neurological injury in a patient with atypical
hemolytic uremic syndrome. Ann Hematol. 2013;
92(4):557-558.
4. Cataland SR, Yang S, Wu HM. The use of
ADAMTS13 activity, platelet count, and serum
creatinine to differentiate acquired thrombotic
thrombocytopenic purpura from other thrombotic
microangiopathies. Br J Haematol. 2012;157(4):
501-503.
5. Coppo P, Schwarzinger M, Buffet M, et al;
French Reference Center for Thrombotic
Microangiopathies. Predictive features of severe
acquired ADAMTS13 deficiency in idiopathic
thrombotic microangiopathies: the French TMA
reference center experience. PLoS ONE. 2010;
5(4):e10208.
6. Wu TC, Yang S, Haven S, et al. Complement
activation and mortality during an acute episode of
thrombotic thrombocytopenic purpura. J Thromb
Haemost. 2013;11(10):1925-1927.
7. Cataland SR, Wu HM. Atypical hemolytic uremic
syndrome and thrombotic thrombocytopenic
purpura: clinically differentiating the thrombotic
microangiopathies. Eur J Intern Med. 2013;24(6):
486-491.
8. Jin M, Casper TC, Cataland SR, et al.
Relationship between ADAMTS13 activity in
clinical remission and the risk of TTP relapse. Br J
Haematol. 2008;141(5):651-658.
9. Caprioli J, Noris M, Brioschi S, et al; International
Registry of Recurrent and Familial HUS/TTP.
Genetics of HUS: the impact of MCP, CFH, and IF
mutations on clinical presentation, response to
treatment, and outcome. Blood. 2006;108(4):
1267-1279.
10. Noris M, Remuzzi G. Atypical hemolytic-uremic
syndrome. N Engl J Med. 2009;361(17):1676-1687.
11. Réti M, Farkas P, Csuka D, et al. Complement
activation in thrombotic thrombocytopenic
purpura. J Thromb Haemost. 2012;10(5):791-798.
12. Harboe M, Thorgersen EB, Mollnes TE. Advances
in assay of complement function and activation.
Adv Drug Deliv Rev. 2011;63(12):976-987.
13. Lotta LA, Lombardi R, Mariani M, et al. Platelet
reactive conformation and multimeric pattern of
von Willebrand factor in acquired thrombotic
thrombocytopenic purpura during acute disease
and remission. J Thromb Haemost. 2011;9(9):
1744-1751.
14. Moake JL, Rudy CK, Troll JH, et al. Unusually
large plasma factor VIII: von Willebrand factor
multimers in chronic relapsing thrombotic
thrombocytopenic purpura. N Engl J Med. 1982;
307(23):1432-1435.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2014 123: 3733-3738
doi:10.1182/blood-2013-12-547067 originally published
online April 2, 2014
Biomarkers of terminal complement activation confirm the diagnosis of
aHUS and differentiate aHUS from TTP
Spero R. Cataland, V. Michael Holers, Susan Geyer, Shangbin Yang and Haifeng M. Wu
Updated information and services can be found at:
http://www.bloodjournal.org/content/123/24/3733.full.html
Articles on similar topics can be found in the following Blood collections
Clinical Trials and Observations (4553 articles)
Free Research Articles (4527 articles)
Platelets and Thrombopoiesis (729 articles)
Red Cells, Iron, and Erythropoiesis (794 articles)
Thrombocytopenia (231 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.